HIV-1/2 Antigen and Antibodies, Fourth Generation, with Reflexes

Test code(s) 91431(X)

Question 1. Are these particular tests appropriate for use in infants below the age of 2 years?

No. The HIV antigen/antibody assay listed above detects HIV antibodies that could be passively transferred from infected mothers to their infants. The appropriate tests to aid in the diagnosis of infant HIV infection include1,10:

HIV-1 RNA, Qualitative TMA (test code16185[X])

HIV-1 DNA, Qualitative PCR (test code 8401[X])

HIV-2 DNA-RNA, Qualitative RT-PCR (test code 34977[X])

Question 2. Who should be screened for the presence of HIV infection?

The latest (2006) Centers for Disease Control and Prevention (CDC) recommendations for HIV testing of individuals encountered in healthcare settings include2:

Routine, rather than behavior risk-based, HIV screening for all patients aged 13-64 years unless the prevalence of infection is extremely low (<1 in 1000 persons)

Opt-out testing (ie, HIV testing performed unless patient declines)

Persons engaging in risk behaviors associated with HIV acquisition should be screened for HIV infection at least annually

The US Preventive Services Task Force (USPSTF) recommends clinicians screen for HIV infection in adolescents and adults aged 15 to 65. Younger adolescents and older adults who are at increased risk should also be screened.3

HIV antigen/antibody fourth-generation-based screening tests can simultaneously detect both HIV-1 antigen (ie, viral protein) and HIV-1/2 antibodies, whereas HIV-1/2 antibody third-generation-based screening tests detect only antibodies. Third-generation test results are considered repeatedly reactive only after a person has developed detectable levels of antibody in their serum or other body fluid. Because third-generation tests detect only antibodies to HIV, they cannot detect HIV infection during acute infection—the period after virus acquisition when viral protein (HIV-1 antigen) may be detectable but antibodies to HIV are not.4 In contrast, fourth-generation HIV-1/2 testing methods can detect HIV-1 antigen as well as HIV-1 and HIV-2 antibodies.

Thus fourth-generation-based screening tests can detect HIV during a portion of the acute phase and all of the chronic phase of the disease. This ability has substantial individual and societal benefits. The patient benefits in that he/she can be more quickly evaluated for administration of appropriate antiviral medications, which may reduce the adverse early impacts of infection. Society benefits because actions can be taken to reduce HIV transmission. Individuals with acute HIV infection have much higher viral loads, making them more likely to transmit the virus. However, if individuals are diagnosed during the acute phase of the illness, they can be counseled regarding risk-reduction practices and can reduce their viral load with antiviral medication. Both of these actions reduce the likelihood of transmission to uninfected partners.

The CDC recommends using a fourth generation HIV antigen/antibody test for screening and diagnosis because it can detect HIV during both the acute and chronic phases of infection.5

Question 4. How long after exposure can fourth-generation tests effectively rule out HIV infection?

A study using a variety of HIV-1 seroconversion sample panels, in conjunction with statistical modeling, estimated that half of patients become repeatedly reactive on fourth-generation testing within 18 days after HIV infection; more than 99% have reactive results within 45 days after HIV acquisition. Therefore, HIV infection is very unlikely (ie, less than 1%) in patients with negative fourth-generation test results at least 45 days after the most recent exposure.11

Question 5. What is the CDC-recommended testing algorithm for the diagnosis of HIV infection?

The key concepts associated with the 2014 CDC recommendations are illustrated here.5

Question 6. Why does the CDC no longer recommend the HIV-1 Western blot test as the supplemental antibody test?

The CDC no longer recommends using the Western blot technique to confirm the presence of HIV-1 antibodies in repeatedly reactive specimens because:

The HIV-1/HIV-2 antibody differentiation test is more sensitive than the Western blot early in infection.6

The HIV-1/HIV-2 antibody differentiation test can be performed more rapidly than can the Western blot method, so the turnaround time for confirmation of HIV infection is shorter.

Studies have shown that the HIV-1/HIV-2 antibody differentiation test more accurately differentiates HIV-1 and HIV-2 infections.7 HIV-2 infection, while rare in the United States, is most commonly seen in persons from West Africa. It's important to differentiate HIV-1 from HIV-2, as HIV-2 may not respond to some HIV medications.9

Question 7. How is the following combination of HIV test results interpreted?

A patient with these test results…

A patient with these test results…

…has acute HIV-1 infection, confirmed by detection of HIV-1 RNA prior to the development of HIV-1 antibodies.

The precise reason for a biological false-positive result in any individual specimen is not likely to be definitively known. A few of the causes hypothesized in the medical literature include recent administration of selected vaccines, presence of HLA-DR antibodies, presence of rheumatoid factors, reactive RPR, hypergammaglobulinemia, cross-reactive immune response to other exogenous and endogenous retroviruses, and autoimmune conditions.

Although there are occasional biological false-positive results observed with HIV screening tests, as with all screening tests, the specificities of the FDA-approved HIV screening tests are very high (>99%). The potential adverse effects of a biological false-positive screening test result are ameliorated by use of this CDC-recommended multi-step testing algorithm. Test results are only considered to support the diagnosis of HIV infection when the screening test results are repeatedly reactive and either supplemental test (ie, HIV-1/2 antibody differentiation or HIV-1 RNA) is positive.5

Question 9. Which follow-up test is recommended when the screening test is Repeatedly Reactive, but the confirmatory tests are both negative?

Follow-up testing is generally not recommended. Additional testing is only indicated if the patient is thought to be very recently infected and/or the patient is at continued risk for HIV exposure. Refer to cdc.govfor the latest CDC recommendations on this subject.

There have been anecdotal reports of pregnancy being associated with a higher frequency of biological false-positive HIV screening test results. This anecdotal association may have been due to an initially greater frequency of testing of pregnant women prior to issuance of the 2006 CDC HIV testing recommendations. A more recent large epidemiologic study using a third-generation HIV screening method did not demonstrate a greater propensity toward biological false positivity in pregnant subjects. In this study of more than 2 million third-generation HIV antibody screening tests, the false-positive rate was actually lower for pregnant women than for others (0.14% versus 0.21%).8

Question 11. Why does the laboratory require that a blood sample for HIV testing be collected in a separate tube?

HIV screening tests are extremely sensitive and can detect very low levels of circulating viral antibodies and/or antigens (fourth-generation test) in a patient’s sample. The laboratory requires a separate sample collection tube for HIV screening testing to help eliminate false-positive results that may be associated with pre-analytic sample handling during the performance of other requested tests. Minimizing pre-analytic sample handling issues is critical, given the clinical significance of a positive HIV test result.

Question 12. What percentage of patient specimens tested with the fourth-generation-based HIV diagnostic algorithm require being reflexed through the entire algorithm?

A retrospective unpublished evaluation of approximately 23,000 samples tested at Quest Diagnostics using the fourth-generation HIV screening algorithm found that only 0.16% required reflex testing through the final step in the CDC HIV diagnostic algorithm (ie, testing by the HIV-1 RNA assay).

Branson BM, Owen SM, Wesolowski LG, et al for Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations. http://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014. Accessed June 27, 2014.

This FAQ is provided for informational purposes only and is not intended as medical advice. A clinician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.